Activated sludge (AS) from wastewater treatment plant of brewery industry was used as substrate for hydrogen production by anaerobic mixed cultures in batch fermentation process. The AS (10% TS) was pretreated by acid, heat and combined acid and heat. Combined acid- heat treatment (0.5% (w/v) HCl, 110 degrees C, 60 min) gave the highest soluble COD (sCOD) of 1785.6 +/- 27.1 mg/L with the highest soluble protein and carbohydrate of 8.1 +/- 0.1 and 38.5 +/- 0.8 mg/L, respectively. After the pretreatment, the pretreated sludge was used to produce hydrogen by heat treated upflow anaerobic sludge blanket (UASB) granules. A maximum hydrogen production potential of 481 mL H-2/L was achieved from the AS pretreated with acid (0.5% (w/v) HCl) for 6 h. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.